草鱼Ⅲ型呼肠孤病毒外衣壳蛋白VP38的表达分析及多克隆抗体制备

Expression Analysis and Polyclonal Antibody Preparation of Outer Shell Protein VP38 Encoded by Genotype Ⅲ Grass Carp Reovirus

为进一步开展草鱼Ⅲ型呼肠孤病毒外衣壳蛋白VP38的生物学功能研究准备实验材料,同时探讨并构建一种草鱼Ⅲ型呼肠孤病毒的免疫学检测方法。实验构建了VP38的原核表达质粒pET28a-VP38,转化至BL21感受态细胞后利用IPTG(Isopropyl β-D-Thiogalactoside)诱导表达,8 mol/L尿素溶解重组蛋白后免疫小鼠,制备鼠抗VP38多克隆抗体;利用制备的抗体探究Grass carp reovirus 104(GCRV-104)感染后VP38在翻译水平的表达动力学;利用Western blot、间接免疫荧光分析(IFA)对抗体进行评估;构建VP38的真核表达载体pEGFP-N1-VP38,转染至草鱼性腺(Grass carp ovary,GCO)细胞内进行亚细胞定位分析。结果显示:重组VP38蛋白在原核表达系统中以包涵体形式存在;制备的鼠抗VP38多克隆抗体既能够识别重组VP38蛋白,也能够识别GCO细胞感染GCRV-104后表达的VP38蛋白;GCRV-104感染后72h VP38主要分布在细胞质中,与亚细胞定位结果一致;VP38在感染的前期微量表达,感染中后期大量表达。本研究制备的鼠抗VP38多克隆抗体具有较高的效价和较好的特异性,为构建草鱼Ⅲ型呼肠孤病毒的免疫学检测方法提供了较好的技术路线。

To prepare experimental materials for further research on the biological function of VP38 and build an immunological detection method of genotype Ⅲ grass carp reovirus,in the experiment the prokaryotic expression plasmid pET28a VP38 was constructed and transformed into BL21 competent cells,the expression of recombinant fusion protein was induced by IPTG and dissolved in 8 mol/L urea,the mice were immunized with recombinant protein to prepare mouse anti-VP38 polyclonal antibodies.Western blot and indirect immune fluorescence analysis(IFA)were carried out to evaluate the antibody.The expression kinetics of VP38 at the translation level after GCRV-104 infection was investigated with the prepared antibodies.The antibody was evaluated by Western blot and indirect immunofluorescence assay(IFA).The eukaryotic expression vector pEGFP-N1-VP38 was constructed and transfected into grass carp GCO cells for sub cellular localization analysis.The results showed that the recombinant VP38 protein existed as an inclusion body in the prokaryotic expression system.The prepared mouse anti VP38 polyclonal antibody could recognize both the recombinant VP38 protein and the VP38 protein expressed by GCO cells infected with GCRV-104.VP38 was mainly distributed in the cytoplasm 72 h after GCRV-104 infection,which was consistent with the results of sub cellular localization.VP38 was expressed in a small amount in the early stage of infection and in a large amount in the middle and late stage of infection.The mouse anti VP38 polyclonal antibody prepared in this study has higher titer and better specificity,and provides a better technical route for constructing the immunological detection method of genotype Ⅲ grass carp reovirus.